The effects of IL-1β stimulated human umbilical cord mesenchymal stem cells on polarization and apoptosis of macrophages in rheumatoid arthritis

Macrophages play an important role in the pathogenesis of rheumatoid arthritis (RA), in which the functions of pro-inflammatory macrophages (M1) and anti-inflammatory macrophages (M2) are different. Our previous studies have demonstrated that interleukin-1β (IL-1β) stimulated human umbilical cord mesenchymal stem cells (hUCMSCs) increase the expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and initiate breast cancer cell apoptosis via ligand to death receptor 4 (DR4) and DR5. In this study, we examined the effect of IL-1β stimulated hUCMSCs (IL-1β-hUCMSCs) on immunoregulation of M1 and M2 macrophages in vitro and in the RA mouse model. The results showed that IL-1β-hUCMSCs increased macrophage polarization into M2 macrophages and enhanced apoptosis of M1 macrophages in vitro. Moreover, the intravenous injected IL-1β-hUCMSCs in RA mice rehabilitated the imbalance of M1/M2 ratio and thus demonstrated the potential to reduce inflammation in RA. This study advances our knowledge of the underlying immunoregulatory mechanisms involved in IL-1β-hUCMSCs to induce M1 macrophage apoptosis and promote the anti-inflammatory polarization of M2 macrophages and demonstrates the potential of IL-1β-hUCMSCs to reduce inflammation in RA.


Cytotoxic impact of LPS and IL-4 on Raw264.7 cells and KG-1 cells.
To examine the cytotoxicity of Lipopolysaccharide (LPS) and Interleukin-4 (IL-4), Raw264.7 cells and KG-1 cells were treated with 0 to 1 μg/ mL LPS and 0 to 100 ng/mL IL-4 for 24 h and detected by MTT assay. The results showed that the different concentrations of LPS and IL-4 used in these experiments had no significant cytotoxic effects on these two cell lines ( Fig. 1a-d). Therefore, the concentration of 0.025, 0.5, 1 μg/mL LPS and 5, 10, 20 ng/mL IL-4 were used for the subsequent macrophage polarization experiments.
The effects of LPS and IL-4 in macrophages polarization, DR4 and DR5 expression. Western blot and flow cytometry were used to examine whether LPS and IL-4 induced Raw264.7 cells and KG-1 cells polarized into M1 macrophages or M2 macrophages. After treatment with LPS or IL-4, the expression of DR4 and DR5 in both cell lines were also detected. The Western blot data showed that treatment with 0.5 and 1 μg/mL LPS significantly increased the expression of iNOS in Raw264.7 cells (Fig. 2a) and in KG-1 cells (Fig. 2c). DR4 and DR5 expression only increased in LPS induced Raw264.7 cells. DR4 expression increased after treatment with 0.5 and 1 μg/mL LPS and DR5 increased after treatment with 0.025 μg/mL LPS (Fig. 2a). The results indicated that macrophages with LPS treatment were successfully polarized to M1 macrophages and DR4 and DR5 expressions were higher in these M1-polarized macrophages. Treatment with 10 and 20 ng/mL IL-4 decreased the expression of iNOS in Raw 264.7 cells (Fig. 2b) and KG-1 cells (Fig. 2d). The expression of DR5 was decreased in both 20 ng/mL IL-4 induced Raw264.7 cells (Fig. 2c) and KG-1 cells (Fig. 2d).
The M2 phenotype status of LPS and IL-4 induced Raw 264.7 cells and KG-1 cells were characterized by CD163 expression via flow cytometry. The results showed that the expression of CD163 in Raw 264.7 cells and KG-1 did not increase after being treated with LPS (Fig. 2e,f). However, the expression of CD163 significantly increased in 5, 10, and 20 ng/mL IL-4 stimulated Raw 264.7 cells (Fig. 2e) and in 20 ng/mL IL-4-stimulated KG-1 cells (Fig. 2f). According to the above experimental data, 1 μg/mL LPS and 20 ng/mL IL-4 were chosen for use in the subsequent experiments. These results demonstrated that LPS could induce M1 polarization and IL-4 could successfully induce M2 polarization in Raw 264.7 cells and KG-1 cells. Moreover, TRAIL receptors DR4 and DR5 expression significantly increased in LPS induced M1 polarized Raw 264.7 cells. DR5 expression was decreased in IL-4 treated Raw 264.7 cells and KG-1 cells, suggesting that DR5 expression was decreased in IL-4 induced M2 polarization in Raw 264.7 cells and KG-1 cells.
The effect of IL-1β-hUCMSCs on macrophages polarization. Macrophage infiltration is one of the symptoms in rheumatoid arthritis 27 . A large number of M1 macrophages secrete pro-inflammatory factors, which not only aggravates the disease but also makes more M0 macrophages polarized into M1 macrophages. www.nature.com/scientificreports/ To know whether IL-1β-hUCMSCs could induce macrophage polarization, we co-cultured macrophages with IL-1β-hUCMSCs directly or indirectly. Flow cytometry data showed that the expression of iNOS was not statistically significantly different in Raw264.7 and KG-1 cells directly or indirectly co-cultured with IL-1β-hUCMSCs. However, the expression of CD163 in both Raw264.7 and KG-1 cells significantly increased after direct and indirect co-culture with IL-1β-hUCMSCs ( Fig. 3a,b). The results show that Raw 264.7 cells and KG-1 cells cocultured with IL-1β-hUCMSCs did not increase M1 macrophages polarization but increased M2 macrophages polarization.
IL-1β-hUCMSCs induce the apoptosis of M1 macrophage. Aggregation of M1 macrophages have been found to promote inflammation in rheumatoid arthritis 33 . Therefore, we wanted to know the effects of IL-1β-hUCMSCs on apoptosis of M1 macrophages. Raw264.7 cells and KG-1 cells were induced into M1 and M2 macrophages then directly or indirectly co-cultured with IL-1β-hUCMSCs. Flow cytometry data showed that the apoptotic rate of M1-polarized Raw264.7 cells was increased after direct and indirect co-culture with IL-1β-hUCMSCs (Fig. 4a), while the apoptotic rate of M2-polarized Raw264.7 cells was decreased (Fig. 4a). The apoptotic rate of M1-polarized KG-1 cells significantly increased after indirect co-cultured with IL-1β-hUCMSCs (Fig. 4b). However, the apoptotic rate of M2-polarized Raw264.7 cells and KG-1 cells decreased after indirect co-culture with IL-1β-hUCMSCs (Fig. 4a,b). These data suggested that IL-1β-hUCMSCs could enhance the apoptosis of M1 macrophages and inhibit the apoptosis of M2 macrophages.
The effects of IL-1β-hUCMSCs on macrophage proliferation. We had demonstrated that the apoptotic rate of M1 macrophages increased after co-culture with IL-1β-hUCMSCs. Next, we examine the effect of IL-1β-hUCMSCs on M1 and M2 macrophage proliferation. The results of flow cytometry showed that IL-1β-hUCMSCs had no effect on the proliferation of M1-polarized Raw246.7 cells. But increased M2-polarized Raw246.7 cells proliferation after indirect co-culture with IL-1β-hUCMSCs (Fig. 6a). While the proliferation rate of M1-polarized and M2-polarized KG-1 cells decreased after co-culture with IL-1β-hUCMSCs (Fig. 6b). According to the above results, the effects of IL-1β-hUCMSCs on macrophage proliferation might be related to increased M1 macrophage cell apoptosis and M2 macrophage proliferation.
The ratio of M1 and M2 macrophages in synovial tissues of CIA mice after intravenous injection of IL-1β-hUCMSCs. The effect of IL-1β-hUCMSCs in the CIA mouse model was examined. Mice were and KG-1 cells were treated 1 µg/mL LPS or 20 ng/mL IL-4 for 24 h to induce into M1 or M2 macrophages, then co-cultured with IL-1β-hUCMSCs for 24 h. After co-culturing, cells were labeled with CD105 antibody to distinguish hUCMSCs from macrophages and stained with Annexin V-FITC and PI to analyze the apoptosis of (a) Raw264.7 cells and (b) KG-1 cells. Statistical analysis data was analyzed by one-way ANOVA with Tukey's test. Data are shown as mean ± SEM (n = 3, *P < 0.05, **P < 0.01, ***P < 0.001). www.nature.com/scientificreports/ induced to develop RA then injected with IL-β-hUCMSCs by intravenous injection and sacrificed after 20 days to obtain the articulating tissue. Immunohistochemical staining was performed to examine the presence of M1 and M2 macrophages in the joint sections. The results showed that the RA group had more M1 macrophages, but the number of M1 macrophages decreased after injection of IL-1β-hUCMSCs (Fig. 7a). However, compared with the MSC group, more M2 macrophages can be found in the sections of the IL-1β-hUCMSCs group (Fig. 7b). By analyzing the M1/M2 ratio, we found that the ratio was 1.9 in the RA group, 1.2 in hUCMSCs group, and the ratio was decreased to 0.7 in IL-1β-hUCMSCs group. This result demonstrates the significant effect of IL-1β-hUCMSCs treatment in modulating M2 macrophage polarization as compared to using hUCMSCs alone.

Discussion
Rheumatoid arthritis is a chronic systemic autoimmune disease that affects the joints of the patients. Monocytes and macrophages are at the core of RA inflammation, and the expression of inflammatory M1 macrophages is increased in RA 35 . Even though there are many RA treatments available, the therapies for RA sometimes fail or are only partially successful 36 . Therefore, in recent years, the prevention or reduction of inflammatory macrophages to alleviate symptoms is now part of the research direction, as a new target therapy in RA.
To ameliorate RA, some macrophage-targeted treatments have been applied, however, these therapies do not specifically target pro-inflammatory macrophages. A strategy for RA therapy would be to develop treatments that prevent the reduction of anti-inflammatory macrophages or aim at impeding the hyper-infiltration of pro-inflammatory macrophages in the RA joint synovium. Lipopolysaccharide (LPS) can induce macrophage polarization to the M1 macrophages and interleukin 4 (IL-4) can drive macrophage polarization to M2 macrophages 37 . In this Figure 5. IL-1β pre-stimulated hUCMSCs induce M1 macrophages to utilize extrinsic and intrinsic pathways for apoptosis (a) Raw264.7 cells and (b) KG-1 cells were induced by 1 μg/mL LPS and 20 ng/mL IL-4 to M1 macrophages and M2 macrophages, and then co-cultured with IL-1β-hUCMSCs for 24 h. The expression of cleaved caspase-3, -8 and -9 was detected by western blot and quantified using AlphaEaseFC 4.0 software. The full-length Western blots were shown in Supplementary Fig. S1. Statistical analysis data was analyzed by oneway ANOVA with Tukey's test. Data are shown as mean ± SEM (n = 3, *P < 0.05, **P < 0.01, ***P < 0.001). www.nature.com/scientificreports/ study, we used LPS and IL-4 to induce macrophage to M1 and M2 macrophages to investigate the mechanisms underlying the therapeutic effects of IL-1β-hUCMSCs on macrophage polarization and macrophage apoptosis in rheumatoid arthritis. TRAIL is a pro-apoptotic protein that can induce malignant cell death, but different cells have different sensitivity to TRAIL, making it difficult to use TRAIL as a therapy. In our previous study, we found that both membrane bound TRAIL and soluble TRAIL expression in hUCMSCs can be upregulated after IL-1β stimulation. By binding to DR4 and DR5, TRAIL initiates apoptotic pathway 38 . Many cytokines and drugs can enhance TRAIL-mediated apoptosis by upregulating DR4 and DR5 expressions. In this study, we found that the increase of TRAIL in IL-1β-hUCMSCs can enhance M1 macrophages apoptosis (Fig. 4) via the up-regulation of cleaved caspase-3, cleaved caspase-8, and cleaved caspase-9, which confirms that TRAIL triggers M1 macrophage apoptosis through extrinsic and intrinsic pathways (Fig. 5).
In rheumatoid arthritis, the polarization of macrophages plays a crucial role. M1 macrophages produce pro-inflammatory factors, aggravate the symptoms of RA, and cause joint and periarticular damage, while M2 macrophages release anti-inflammatory factors to repair the symptoms of RA 39,40 . Because of the different underlying functions of M1 and M2 macrophages, it is best to reduce M1 macrophages or increase M2 macrophages to ameliorate RA symptoms. The application of monoclonal antibodies against macrophage-colony stimulating factor (M-CSF) has been found to reduce the number of monocytes in RA patients 37 . Sinomenine, the main active ingredient of the plant Sinomenium acutum, can regulate the secretion of inflammatory cytokines and monocyte or macrophage subsets 41 . However, the target and underlying mechanism of sinomenine to suppress RA progression has not been elucidated. In our experiments, we demonstrated that IL-1β-hUCMSCs could slow the proliferation of M1 macrophages (Fig. 6), and IL-1β-hUCMSCs could also increase the rate of macrophage polarized into M2 macrophages. The summarized results in Table 1 show that the apoptosis rate was increased under direct and indirect co-culture with IL-1β-hUCMSCs. M1 macrophages indirectly co-cultured with IL-1β-hUCMSCs showed more impact in increasing the apoptosis rates and the expression levels of cleaved caspase-3, -8, -9 (Table 1). From our previous study, we found that both soluble and membrane bound TRAIL www.nature.com/scientificreports/ can be induced by IL-1β stimulation 42 . Thus, the action of soluble TRAIL present in the indirect co-culture conditioned medium may be more effective in inducing M1 macrophage apoptosis. On the contrary, the proliferation of M1 and M2 macrophages decreased in direct co-culture conditions in comparison with indirect co-culture with IL-1β-hUCMSC (Table 1). This effect may involve cell-to-cell contact between macrophages and IL-1β-hUCMSCs, leading to direct interactions and signal transductions that could have a more pronounced effect on macrophage proliferation. According to our results, after successfully inducing macrophages into M1 macrophages and M2 macrophages, TRAIL secreted by IL-1β-hUCMSCs can induce apoptosis of M1 macrophages through both internal and external pathways and reduce their ability to proliferate. In addition, IL-1β-hUCMSCs promote M2 macrophage polarization. In RA synovial tissues, the proportion of pro-inflammatory M1 macrophages is higher than anti-inflammatory M2 macrophages. Targeting M1 macrophages and enhancing the polarization of M2 macrophages in RA to rebalance the M1/M2 ratio would be suitable for RA therapy. MSCs have been used as a promising therapy for immune-related diseases 43,44 . Results from an in vivo mouse model demonstrated that synovial M1 macrophages were reduced and M2 macrophages were increased after injection of IL-1β-hUCMSCs www.nature.com/scientificreports/ (Fig. 7). Compared with the hUCMSCs group, the M1/M2 ratio of the IL-1β-hUCMSCs group supports IL-1β-hUCMSCs as a potential therapeutic strategy. The results of this in vivo study are consistent with the in vitro data that IL-1β-hUCMSCs induce apoptosis of M1 macrophages via TRAIL/DR4 or TRAIL/DR5 interaction and enhance M2 macrophage polarization. In summary, this study indicates that the expression of DR4 and DR5 were upregulated in M1 macrophages. TRAIL expression in hUCMSCs was enhanced after IL-1β stimulation. Via TRAIL/DR4/5 interaction, IL-1β-hUCMSCs induce the apoptosis of M1 macrophages. Furthermore, IL-1β-hUCMSCs enhance the polarization of M2 macrophages (Fig. 8).
We conclude that this study advances our understanding of the immunoregulatory mechanisms involved in intravenous injected IL-1β-hUCMSCs in a RA mice model and hints at the potential of hUCMSCs for RA therapy. However, several limitations in this study that could be addressed in future research include determining the mechanisms and signaling pathways involved in IL-1β-hUCMSCs enhanced M2 polarization. Whether the rehabilitated M1/M2 ratio could affect the synovial hyperplasia in RA synovial intima is also worthy of investigation. The effects of IL-1β-hUCMSCs in this RA model were observed after administration of injections for 20 days. Thus, the issues of long-term efficacy and safety of IL-1β-hUCMSCs treatment in RA therapy still needs to be studied.

Co-culture system of macrophages and IL-1β-hUCMSCs.
For direct co-culture, Raw264.7 and KG-1 cells were plated 2 × 10 5 cells in 30 mm petri dishes with culture medium for 24 h and then starved in serum-free medium for 16 h. After starvation, cells were treated with 1 μg/mL LPS (polarization into M1 macrophages) and 20 ng/mL IL-4 (polarization into M2 macrophages) for 24 h. hUCMSc were seeded 1 × 10 5 cells in 60 mm petri dishes for 24 h. Then starved in serum-free DMEM-LG medium for 16 h and treated with 100 ng/mL IL-1β for 24 h. After pre-treatment of macrophage cell lines and hUCMSCs, both were detached with Accutase ®and seeded into the same 30 mm well for co-culture. For indirect co-culture, Raw264.7 and KG-1 cells were plated 2 × 10 5 cells in 6 well plates with culture medium for 24 h and starved in serum-free medium for 16 h. After starvation, cells were treated with 1 μg/mL LPS (polarization into M1) or 20 ng/mL IL-4 (polarization into M2) for 24 h. hUCMSCs were seeded 1 × 10 5 cells in 0.4 μm pore, 24 mm transwell inserts with culture medium for 24 h. Next, cells were starved in serum-free DMEM-LG for 16 h then treated with 100 ng/mL IL-1β for 24 h. After pre-treatment of macrophages and hUCMSCs, the transwell inserts with hUCMSCs were moved into 6 well plates to co-culture with Raw264. Arthritis developed on day 35 after first injection, then, PBS, hUCMSCs or IL-1β-hUCMSCs were used for treatment by tail vein injection. Animals were anesthetized with Isoflurane on the indicated days and sacrificed by cervical detachment. The limbs of the mice were removed and handed over to Bio-Check Laboratories LTD (Taipei, Taiwan) to be embedded in wax and sectioned at a thickness of 3 μm.
Immunohistochemistry assay. The sections of joints were deparaffinized with xylene and rehydrated using different percentages of ethanol. After rehydration, cells were washed with ddH2O to remove ethanol. Slides were blocked with endogenous peroxidase with 3% H 2 O 2 for 15 min, then washed with PBST three times for 10 min. For antigen retrieval, the slides were placed in the glass staining tank and soaked in a sodium citrate buffer for 10 min at 95 °C. Then the glass staining tank was removed from the heater and the solution cooled down to room temperature (RT). The slides were washed with PBST three times for 10 min. After blocking with 5% Fish Gelatin Buffer (AMRESCO, OH, USA) and 0.1% triton X-100 in PBST (blocking Buffer) for 60 min at RT, the cells were then incubated with iNOS antibody (Abcam, Boston, USA) diluted at 1:100 and CD163 antibody (Abcam, Boston, USA) diluted at 1:100 at 4 °C overnight. The slides were washed with PBST for 10 min three times and incubated with Rabbit IgG antibody (HRP) (GeneTex, CA, USA) for one hour at RT. After incubation, cells were washed with PBST for 5 min three times, then the specimen was covered with DAB solution for 20 min. The slides were washed with H 2 O and counterstained with hematoxylin for 60 s. Finally, the slides were dehydrated with different concentrations of ethanol and ethanol was cleared with xylene. Then a mounting medium was used to mount coverslips over the specimen. The M1 and M2 macrophages of each group were counted in 3 different tissue sections. The cell counting results were analyzed and plotted with Prism 8.0 software. Statistical analysis. Quantitation data were represented as mean ± SEM. Statistical analysis data was analyzed by one-way ANOVA. P values < 0.05 were considered as statistically significant by Dunnett's test and Tukey's test. All quantitative analysis data were performed with Prism 6.0 software. Ethics approval and consent to participate. All